Title: Functional Role of the Third Cytoplasmic Loop in Muscarinic Receptor Dimerization
Abstract: By means of the expression of two chimeric receptors, α2/m3 and m3/α2, in which the carboxyl-terminal receptor portions, containing transmembrane (TM) domains VI and VII, were exchanged between the α2C adrenergic and the m3 muscarinic receptor, Maggio et al. (Maggio, R., Vogel, Z., and Wess, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3103-31073) demonstrated that G protein-linked receptors are able to interact functionally with each other at the molecular level to form (hetero)dimers. In the present study we tested the hypothesis that interaction between receptors might depend on the presence of a long third intracellular (i3) loop and that shortening this loop could impair the capability of receptors to form dimers. To address this question, we initially created short chimeric α2 adrenergic/m3 muscarinic receptors in which 196 amino acids were deleted from the i3 loop (α2/m3-short and m3/α2-short). Although co-transfection of α2/m3 and m3/α2 resulted in the appearance of specific binding, the co-expression of the two short constructs (α2/m3-short and m3/α2-short), either together or in combination, respectively, with m3/α2 and α2/m3 did not result in any detectable binding activity. In another set of experiments, a mutant m3 receptor, m3/m2(16aa), containing 16 amino acids of the m2 receptor sequence at the amino terminus of the third cytoplasmic loop, which was capable of binding muscarinic ligands but was virtually unable to stimulate phosphatidylinositol hydrolysis, was also mutated in the i3 loop, resulting in the m3/m2(16aa)-short receptor. Although co-transfection of m3/m2(16aa) with a truncated form of the m3 receptor (m3-trunc, containing an in frame stop codon after amino acid codon 272 of the rat m3 sequence) resulted in a considerable carbachol-stimulated phosphatidylinositol breakdown, the co-transfection of m3/m2(16aa)-short with the truncated form of the m3 receptor did not result in any recovery of the functional activity. Thus, these data suggest that intermolecular interaction between muscarinic receptors, involving the exchange of amino-terminal (containing TM domains I-V) and carboxyl-terminal (containing TM domains VI and VII) receptor fragments depends on the presence of a long i3 loop. One may speculate that when alternative forms of receptors with a different length of the i3 loop exist, they could have a different propensity to dimerize. By means of the expression of two chimeric receptors, α2/m3 and m3/α2, in which the carboxyl-terminal receptor portions, containing transmembrane (TM) domains VI and VII, were exchanged between the α2C adrenergic and the m3 muscarinic receptor, Maggio et al. (Maggio, R., Vogel, Z., and Wess, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3103-31073) demonstrated that G protein-linked receptors are able to interact functionally with each other at the molecular level to form (hetero)dimers. In the present study we tested the hypothesis that interaction between receptors might depend on the presence of a long third intracellular (i3) loop and that shortening this loop could impair the capability of receptors to form dimers. To address this question, we initially created short chimeric α2 adrenergic/m3 muscarinic receptors in which 196 amino acids were deleted from the i3 loop (α2/m3-short and m3/α2-short). Although co-transfection of α2/m3 and m3/α2 resulted in the appearance of specific binding, the co-expression of the two short constructs (α2/m3-short and m3/α2-short), either together or in combination, respectively, with m3/α2 and α2/m3 did not result in any detectable binding activity. In another set of experiments, a mutant m3 receptor, m3/m2(16aa), containing 16 amino acids of the m2 receptor sequence at the amino terminus of the third cytoplasmic loop, which was capable of binding muscarinic ligands but was virtually unable to stimulate phosphatidylinositol hydrolysis, was also mutated in the i3 loop, resulting in the m3/m2(16aa)-short receptor. Although co-transfection of m3/m2(16aa) with a truncated form of the m3 receptor (m3-trunc, containing an in frame stop codon after amino acid codon 272 of the rat m3 sequence) resulted in a considerable carbachol-stimulated phosphatidylinositol breakdown, the co-transfection of m3/m2(16aa)-short with the truncated form of the m3 receptor did not result in any recovery of the functional activity. Thus, these data suggest that intermolecular interaction between muscarinic receptors, involving the exchange of amino-terminal (containing TM domains I-V) and carboxyl-terminal (containing TM domains VI and VII) receptor fragments depends on the presence of a long i3 loop. One may speculate that when alternative forms of receptors with a different length of the i3 loop exist, they could have a different propensity to dimerize. INTRODUCTIONTransmembrane receptors recognize and integrate external signals modifying the metabolism or the ionic equilibrium of the cell milieu. Muscarinic receptors belong to the G-protein-coupled class of receptors (2Hulme E.C. Birdsall N.J.M. Buckley N.J. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 633-673Crossref PubMed Scopus (1089) Google Scholar). Molecular cloning studies have revealed the existence of five structurally related muscarinic receptor proteins (m1-m5; Refs. 3Bonner T.I. Buckley N.J. Young A.C. Brann M.R. Science. 1987; 237: 527-532Crossref PubMed Scopus (1216) Google Scholar and 4Bonner T.I. Young A.C. Brann M.R. Buckley N.J. Neuron. 1988; 1: 403-410Abstract Full Text PDF PubMed Scopus (656) Google Scholar). The five muscarinic receptors are predicted to be composed of seven hydrophobic transmembrane domains (TM domains I-VII) 1The abbreviations used are: TMtransmembranei3third intracellularaaamino acidkbkilobasetrunctruncatedIP1inositol monophosphate. connected by alternating cytoplasmic and extracellular loops, an extracellular amino-terminal domain and an intracellular carboxyl-terminal segment. These receptors couple to a varied group of effectors, including membrane-associated phospholipases, adenylate and guanylate cyclases, and ion channels (5Bernheim L. Mathie A. Hille B. Neurobiology. 1992; 89: 9544-9548Google Scholar, 6Conklin B.R. Brann M.R. Buckley N.J. Ma A.L. Bonner T.I. Axelrod J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8698-8702Crossref PubMed Scopus (129) Google Scholar, 7Hu J. El-Fakahani E.E. J. Neurochem. 1993; 61: 578-585Crossref PubMed Scopus (62) Google Scholar, 8Peralta E.G. Ashkenazi A. Winslow J.W. Ramachandran J. Capon D.J. Nature. 1988; 334: 434-437Crossref PubMed Scopus (548) Google Scholar). The third intracellular (i3) loop of these receptors confers specificity for G-protein coupling (9Wess J. Bonner T.I. Dörje F. Brann M.R. Mol. Pharmacol. 1990; 38: 517-523PubMed Google Scholar). Moreover, it has been suggested that this segment of the receptor is involved in the phenomenon of internalization and down-regulation (10Lameh J. Philip M. Sharma Y.K. Moro O. Ramachandran J. Sadée W. J. Biol. Chem. 1992; 267: 13406-13412Abstract Full Text PDF PubMed Google Scholar, 11Maeda S. Lameh J. Mallet W.G. Philip M. Ramachandran J. Sadée W. FEBS Lett. 1990; 269: 386-388Crossref PubMed Scopus (49) Google Scholar, 12Shapiro R.A. Nathanson N.M. Biochemistry. 1989; 28: 8946-8950Crossref PubMed Scopus (58) Google Scholar).In a previous article, Maggio et al. (13Maggio R. Vogel Z. Wess J. FEBS Lett. 1993; 319: 195-200Crossref PubMed Scopus (118) Google Scholar) showed that muscarinic receptors behave structurally in a fashion analogous to two-subunit receptors. When truncated m2 or m3 receptors (containing TM domains I-V) were co-expressed in COS-7 cells with gene fragments coding for the corresponding carboxyl-terminal receptor portions (containing TM domains VI and VII), functional muscarinic receptors with ligand binding properties similar to the wild type receptors were obtained. These results have been confirmed and extended by Schöneberg et al. (14Schöneberg T. Liu J. Wess J. J. Biol. Chem. 1995; 270: 18000-18006Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), who showed that muscarinic receptors consist not only of two but of multiple autonomous folding units.The association of amino-terminal (containing TM domains I-V) and carboxyl-terminal (containing TM domains VI and VII) receptor domains may occur not only intramolecularly but also intermolecularly, thus providing a molecular basis for receptor dimerization. This was demonstrated by creating two chimeric receptor molecules, α2/m3 and m3/α2, in which the carboxyl-terminal receptor domains (including TMDs VI and VII) were exchanged between the α2C adrenergic and the m3 muscarinic receptor (1Maggio R. Vogel Z. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3103-3107Crossref PubMed Scopus (296) Google Scholar). Although transfection of the two chimeric constructs alone into COS-7 cells did not result in any detectable binding activity, co-expression of the two mutant receptors resulted in a significant number of specific binding sites for the muscarinic ligand N-[3H]methylscopolamine and the adrenergic ligand [3H]rauwolscine.Intuitively, the interaction between the two chimeric receptors is due to the exchange of the amino- and carboxyl-terminal receptor fragments held together by the i3 loop. The considerable extension of the i3 loop could free the amino- and carboxyl-terminal receptor domains, promoting intermolecular association. In the present study, we tested this hypothesis by creating several constructs with a short i3 loop.DISCUSSIONDespite the commonly presented models of G-protein-coupled receptors that depict the protein as a closely packed structure, several reports indicate that G-protein-coupled receptors are in fact formed by multiple autonomous folding domains. In 1988 Kobilka et al. (19Kobilka B.K. Kobilka T.S. Daniel K. Regan J.W. Caron M.G. Lefkowitz R.J. Science. 1988; 240: 1310-1316Crossref PubMed Scopus (604) Google Scholar) described that "split"β2 adrenergic receptors co-expressed in Xenopus oocytes bind adrenergic ligands. The same phenomenon was reported more recently by Maggio et al. (13Maggio R. Vogel Z. Wess J. FEBS Lett. 1993; 319: 195-200Crossref PubMed Scopus (118) Google Scholar) for the m3 muscarinic receptor. In subsequent experiments Schöneberg et al. (14Schöneberg T. Liu J. Wess J. J. Biol. Chem. 1995; 270: 18000-18006Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) demonstrated that muscarinic receptors may consist not only of two but of multiple autonomous folding domains. Splitting the m3 muscarinic receptor in all the three intracellular and three extracellular loops, they demonstrated that some of these split receptors have the ability to maintain muscarinic binding and functional activity. Furthermore, they demonstrated that proper intracellular trafficking and plasma membrane insertion does not require the presence of the full-length receptor protein. In fact, even quite short polypeptides that contain only the first two or three TM domains are properly transported to the plasma membrane.In their report, Schöneberg et al. (14Schöneberg T. Liu J. Wess J. J. Biol. Chem. 1995; 270: 18000-18006Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) showed that among the split receptors, only the receptor split in the i3 loop retained wild type affinity for all muscarinic ligands tested, suggesting that the i3 loop does not exert indirect conformational effects on the proper arrangement of the transmembrane receptor core (formed by TMs I-VII). The long extension of the i3 loop may explain why this segment of the protein does not restrain the rest of the receptor in any particular conformation. Moreover, this characteristic of the i3 loop is probably the basis that allows chimeric α2/m3 and m3/α2 receptors to exchange domains leading to (hetero)dimerization (1Maggio R. Vogel Z. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3103-3107Crossref PubMed Scopus (296) Google Scholar).It is reasonable to think that shortening the i3 loop could impair the ability of receptors to interact. In agreement with this hypothesis, the data presented in this article show that chimeric α2 adrenergic/m3 muscarinic receptors with a large deletion in the i3 loop (α2/m3-short and m3/α2-short) are not able to interact anymore. For these short α2 adrenergic/m3 muscarinic receptor constructs we cannot exclude the possibility that the absence of intermolecular interaction could be due to an alteration in proper protein folding. However, this explanation seems to be unlikely, because other short constructs such as m3-short and m3/m2(16aa)-short (which have the same deletion in the i3 loop) do not display any alteration in their ability to fold and to be inserted into the plasma membrane. Rather, it seems that this deletion favors receptor expression in some way, as is suggested by the slightly but consistently higher number of binding sites observed for these two short constructs compared with the analogous receptors bearing the normal i3 loop (m3 wild type and m3/m2(16aa), respectively; Table II). Epitope-tagged receptors could have been constructed to address whether these two receptors are indeed present on the cell surface, but this experiment would not have excluded the possibility of misfolded protein not able to interact even when present on the membrane.In another set of experiments designed to overcome this problem, we used functionally impaired m3 muscarinic receptors. Like m3/m2(16aa), the short form of this receptor (m3/m2(16aa)-short) displays binding affinities similar to the wild type m3 receptor (besides a slight increase in agonist affinity). Nonetheless, the co-transfection of m3/m2(16aa)-short with m3-trunc was unable to restore phosphatidylinositol hydrolysis activity, indicating that the short i3 loop considerably limits the independence of the amino- and carboxyl-terminal fragments of the m3/m2(16aa)-short receptor and, consequently, their ability to interact with foreign receptor domains.Our data lead to the conclusion that the length of the i3 loop plays a critical role in the regulation of receptor dimerization. Although muscarinic receptors physiologically do not have variants with a shorter i3 loop, dopamine receptors do. For example, an alternative splicing in the i3 loop of the dopamine D2 receptor leads to a short and a long form that differ in length by 29 amino acids (20Giros B. Sokoloff P. Martres M.-P. Riou J.-F. Emorine L.J. Schwartz J.-C. Nature. 1989; 342: 923-926Crossref PubMed Scopus (583) Google Scholar). Even though in our experiments we deleted a very large portion of the protein (196 amino acids) to prevent receptor interaction, it is possible that smaller deletions could be equally effective, if the i3 loop is near a borderline length for intermolecular interaction between receptors.It has been demonstrated that the i3 loop of muscarinic receptors is involved in physiological activities like sequestration and down-regulation. For example, Lameh et al. (10Lameh J. Philip M. Sharma Y.K. Moro O. Ramachandran J. Sadée W. J. Biol. Chem. 1992; 267: 13406-13412Abstract Full Text PDF PubMed Google Scholar), using a series of deletion mutants of the i3 loop of the m1 muscarinic receptor, demonstrated that these deletions left the phosphatidylinositol turnover activity unchanged but impaired the ability of the mutated receptors to internalize. Furthermore, they defined a narrow domain in the middle of the i3 loop (apparently maintained in all five muscarinic receptor subtypes), which is likely to be involved in receptor sequestration.Our experiments performed with the short form of the wild type m3 muscarinic receptor (m3-short) demonstrate that the large deletion of the i3 loop leaves the binding characteristics of the receptor, the phosphatidylinositol hydrolysis activity, and the ability of the receptor to internalize unvaried. These data indicate that the large portion of the i3 loop that has been deleted, at least in the rat m3 muscarinic receptor, is not essential for sequestration, but like the mutant receptor m3/m2(16aa)-short, it might prevent receptor dimerization. As the portion of the i3 loop removed contains several residues that are potential sites of phosphorylation (2Hulme E.C. Birdsall N.J.M. Buckley N.J. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 633-673Crossref PubMed Scopus (1089) Google Scholar), and evidence indicates that phosphorylation of the receptors at serine and threonine residues may be involved in the desensitization of G-protein-linked receptors (21Kobilka B. Annu. Rev. Neurosci. 1992; 15: 87-114Crossref PubMed Scopus (313) Google Scholar, 22Richardson R.M. Hosey M.M. J. Biol. Chem. 1992; 267: 22249-22255Abstract Full Text PDF PubMed Google Scholar), it remains to be established whether this deletion of the i3 loop impairs receptor down-regulation, as has been reported by Shapiro and Nathanson (12Shapiro R.A. Nathanson N.M. Biochemistry. 1989; 28: 8946-8950Crossref PubMed Scopus (58) Google Scholar) for the human m1 muscarinic receptor.At the moment we do not know what the physiological role of receptor dimerization is, and only speculations are possible. For example, interaction between different receptor subtypes could lead to stimulation of different G proteins. We have demonstrated that the amino-terminal domain of the m2 muscarinic receptor can efficiently interact with the carboxyl-terminal domain of the m3 receptor and form a hybrid m2/m3 receptor complex. Co-localization of m2 and m3 muscarinic receptor subtypes in the same cells could lead to the formation of an m2/m3 receptor heterodimer, which could stimulate other G proteins than the wild type m2 and m3 receptors. Alternatively, the intermolecular interaction might promote aggregation of receptors and a consequent compartmentalization of second messenger increase. In support of this view, a recent article by Wreggett and Wells (23Wreggett K.A. Wells J.W. J. Biol. Chem. 1995; 270: 22488-22499Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) based on binding studies and gel electrophoresis experiments on solubilized receptors from porcine atria has clearly demonstrated a cooperativity interaction among muscarinic M2 receptors, and all of their data can be accounted for by cooperative effects within a receptor that is at least tetravalent. Further comparative studies between the wild type m3 muscarinic receptor and the mutant m3-short receptor could reveal the physiological role of the dimerization.In conclusion, we have demonstrated that removal of a large fragment of the i3 loop from the m3 muscarinic receptor does not modify its ability to bind ligands, to stimulate phosphatidylinositol hydrolysis, and to internalize; however, the same deletion alters the intermolecular interaction between receptors, showing that receptor dimerization depends on the length of the i3 loop. Based on the high structural homology found among all G-protein-coupled receptors, our findings should be of general importance for the entire class of integral membrane proteins. INTRODUCTIONTransmembrane receptors recognize and integrate external signals modifying the metabolism or the ionic equilibrium of the cell milieu. Muscarinic receptors belong to the G-protein-coupled class of receptors (2Hulme E.C. Birdsall N.J.M. Buckley N.J. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 633-673Crossref PubMed Scopus (1089) Google Scholar). Molecular cloning studies have revealed the existence of five structurally related muscarinic receptor proteins (m1-m5; Refs. 3Bonner T.I. Buckley N.J. Young A.C. Brann M.R. Science. 1987; 237: 527-532Crossref PubMed Scopus (1216) Google Scholar and 4Bonner T.I. Young A.C. Brann M.R. Buckley N.J. Neuron. 1988; 1: 403-410Abstract Full Text PDF PubMed Scopus (656) Google Scholar). The five muscarinic receptors are predicted to be composed of seven hydrophobic transmembrane domains (TM domains I-VII) 1The abbreviations used are: TMtransmembranei3third intracellularaaamino acidkbkilobasetrunctruncatedIP1inositol monophosphate. connected by alternating cytoplasmic and extracellular loops, an extracellular amino-terminal domain and an intracellular carboxyl-terminal segment. These receptors couple to a varied group of effectors, including membrane-associated phospholipases, adenylate and guanylate cyclases, and ion channels (5Bernheim L. Mathie A. Hille B. Neurobiology. 1992; 89: 9544-9548Google Scholar, 6Conklin B.R. Brann M.R. Buckley N.J. Ma A.L. Bonner T.I. Axelrod J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8698-8702Crossref PubMed Scopus (129) Google Scholar, 7Hu J. El-Fakahani E.E. J. Neurochem. 1993; 61: 578-585Crossref PubMed Scopus (62) Google Scholar, 8Peralta E.G. Ashkenazi A. Winslow J.W. Ramachandran J. Capon D.J. Nature. 1988; 334: 434-437Crossref PubMed Scopus (548) Google Scholar). The third intracellular (i3) loop of these receptors confers specificity for G-protein coupling (9Wess J. Bonner T.I. Dörje F. Brann M.R. Mol. Pharmacol. 1990; 38: 517-523PubMed Google Scholar). Moreover, it has been suggested that this segment of the receptor is involved in the phenomenon of internalization and down-regulation (10Lameh J. Philip M. Sharma Y.K. Moro O. Ramachandran J. Sadée W. J. Biol. Chem. 1992; 267: 13406-13412Abstract Full Text PDF PubMed Google Scholar, 11Maeda S. Lameh J. Mallet W.G. Philip M. Ramachandran J. Sadée W. FEBS Lett. 1990; 269: 386-388Crossref PubMed Scopus (49) Google Scholar, 12Shapiro R.A. Nathanson N.M. Biochemistry. 1989; 28: 8946-8950Crossref PubMed Scopus (58) Google Scholar).In a previous article, Maggio et al. (13Maggio R. Vogel Z. Wess J. FEBS Lett. 1993; 319: 195-200Crossref PubMed Scopus (118) Google Scholar) showed that muscarinic receptors behave structurally in a fashion analogous to two-subunit receptors. When truncated m2 or m3 receptors (containing TM domains I-V) were co-expressed in COS-7 cells with gene fragments coding for the corresponding carboxyl-terminal receptor portions (containing TM domains VI and VII), functional muscarinic receptors with ligand binding properties similar to the wild type receptors were obtained. These results have been confirmed and extended by Schöneberg et al. (14Schöneberg T. Liu J. Wess J. J. Biol. Chem. 1995; 270: 18000-18006Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), who showed that muscarinic receptors consist not only of two but of multiple autonomous folding units.The association of amino-terminal (containing TM domains I-V) and carboxyl-terminal (containing TM domains VI and VII) receptor domains may occur not only intramolecularly but also intermolecularly, thus providing a molecular basis for receptor dimerization. This was demonstrated by creating two chimeric receptor molecules, α2/m3 and m3/α2, in which the carboxyl-terminal receptor domains (including TMDs VI and VII) were exchanged between the α2C adrenergic and the m3 muscarinic receptor (1Maggio R. Vogel Z. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3103-3107Crossref PubMed Scopus (296) Google Scholar). Although transfection of the two chimeric constructs alone into COS-7 cells did not result in any detectable binding activity, co-expression of the two mutant receptors resulted in a significant number of specific binding sites for the muscarinic ligand N-[3H]methylscopolamine and the adrenergic ligand [3H]rauwolscine.Intuitively, the interaction between the two chimeric receptors is due to the exchange of the amino- and carboxyl-terminal receptor fragments held together by the i3 loop. The considerable extension of the i3 loop could free the amino- and carboxyl-terminal receptor domains, promoting intermolecular association. In the present study, we tested this hypothesis by creating several constructs with a short i3 loop.